Composition comprising a gene vector that selectively depletes P16 positive senescent cells

ABSTRACT

Polypeptides, viruses, methods and compositions provided herein are useful for the selective elimination of senescent cells. Method aspects include methods for inducing apoptosis in a senescent cell comprising administering to the cell a polynucleotide, virus, host cell, or pharmaceutical composition described herein. Other methods include expressing a pro-apoptotic gene in a senescent cell comprising administering to the cell the polynucleotide, virus, or pharmaceutical composition as described herein.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.16/029,244, filed Jul. 6, 2018, which is a divisional of U.S. patentapplication Ser. No. 14/394,854, filed Oct. 16, 2014, which is a U.S.371 of International PCT/US2013/036811, filed Apr. 16, 2013, whichclaims the benefit and priority under 35 U.S.C. § 119 (e) to U.S.Provisional Application No. 61/625,612, filed Apr. 17, 2012, the contentof which is incorporated by reference in its entirety into the presentdisclosure.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 44237-716-301-Sequence-Listing.txt. The textfile is 8 KB, and was created on Jun. 7, 2019, and is being submittedelectronically via EFS-Web.

BACKGROUND

Senescent cells accumulate in people as a result of stress, injuries,aging, and related insults leading to a multitude of diseases,conditions and pathologies. The removal of senescent cells couldtherefore provide a dramatic improvement in the quality of life of mosthumans.

Studies using genetically engineered mice have demonstrated that thedestruction of senescent cells in a living organism reverses manydisease-like pathologies. Unfortunately, unique exploitable markers(such as surface epitopes) that can be exploited to design drugs for thespecific killing of senescent cells have not yet been identified. Thus,there is a need in the art for methods and compositions designed tospecifically target and eliminate these cells in vivo. Ideally,elimination of such cells should occur through natural mechanisms suchas apoptosis.

SUMMARY

This invention provides for polynucleotides, viruses, methods andcompositions which are useful for the activation of apoptosis ofsenescent cells. One aspect relates to a polynucleotide comprising apro-apoptotic gene and a p16 promoter, wherein the expression of thepro-apoptotic gene is regulated by the p16 promoter or an equivalentthereof.

A second aspect relates to a virus comprising the polynucleotides asdescribed herein. In one embodiment, the virus is a lytic virus.

Further aspects relate to pharmaceutical compositions comprising thepolynucleotides or virus particles as described herein.

Another aspect relates to a method for expressing a pro-apoptotic genein a senescent cell comprising administering to the cell thepolynucleotide, virus, or pharmaceutical composition as describedherein.

A further aspect relates to a method for inducing apoptosis in asenescent cell comprising administering to the cell the polynucleotide,virus, or pharmaceutical composition as described herein. In certainembodiments, the p16 promoter is activated in the cell. In furtherembodiments, the cell is in vivo in a mammal. In another embodiment, thecell is in vitro.

Another aspect relates to a A conditionally replicating viral constructcomprising an essential viral gene regulated by one or more promoterswherein at least one promoter is the p16 promoter or an equivalentthereof. Also provided, is a method for expressing a conditionallyreplicating virus in a cell comprising infecting the cell with the virusas described herein, wherein the virus replicates in senescent cells butnot in non-senescent cells.

A further method disclosed herein relates to a method for inducingapoptosis in a senescent cell in a subject in need thereof comprisingadministering to the subject a polynucleotide, virus, host cell, orpharmaceutical composition described herein.

Other aspects relate to pharmaceutical compositions comprising thepolynucleotide or virus described herein and a carrier.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows SEQ ID NO: 1, which comprises the human p16 promoter. Thispromoter sequence is further described in Wang et al. “Characterizationof Regulatory Elements on the Promoter Region of p16INK4a Thatcontribute to Overexpression of p16 in Senescent Fibroblasts” J. ofBiol. Chem. (2001), Vol. 276, No. 52, pp. 48655-48661, which is hereinincorporated by reference.

FIG. 2A-B shows SEQ ID NO: 2, which comprises the mouse p16 promoter.

FIG. 3 shows SEQ ID NO: 3, which exemplifies a Caspase 8 polynucleotidesequence from humans.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this disclosure, various technical and patent publicationsare referenced to more fully describe the state of the art to which thisinvention pertains. These publications are incorporated by reference, intheir entirety, into this application.

Definitions

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of tissue culture, immunology,molecular biology, microbiology, cell biology and recombinant DNA, whichare within the skill of the art. See, e.g., Sambrook and Russell eds.(2001) Molecular Cloning: A Laboratory Manual, 3^(rd) edition; theseries Ausubel et al. eds. (2007) Current Protocols in MolecularBiology; the series Methods in Enzymology (Academic Press, Inc., N.Y.);MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press atOxford University Press); MacPherson et al. (1995) PCR 2: A PracticalApproach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual;Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique,5^(th) edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No.4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization;Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds.(1984) Transcription and Translation; Immobilized Cells and Enzymes (IRLPress (1986)); Perbal (1984) A Practical Guide to Molecular Cloning;Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells(Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer andExpression in Mammalian Cells; Mayer and Walker eds. (1987)Immunochemical Methods in Cell and Molecular Biology (Academic Press,London); Herzenberg et al. eds (1996) Weir's Handbook of ExperimentalImmunology; Manipulating the Mouse Embryo: A Laboratory Manual, 3^(rd)edition (Cold Spring Harbor Laboratory Press (2002)).

As used herein, certain terms may have the following defined meanings.

As used in the specification and claims, the singular form “a,” “an” and“the” include plural references unless the context clearly dictatesotherwise.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but do notexclude others. “Consisting essentially of” when used to definecompositions and methods, shall mean excluding other elements of anyessential significance to the combination when used for the intendedpurpose. Thus, a composition consisting essentially of the elements asdefined herein would not exclude trace contaminants or inert carriers.“Consisting of” shall mean excluding more than trace elements of otheringredients and substantial method steps for preparing the microfluidicdevice. Embodiments defined by each of these transition terms are withinthe scope of this invention.

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 0.1. It is to be understood, althoughnot always explicitly stated that all numerical designations arepreceded by the term “about”. It also is to be understood, although notalways explicitly stated, that the reagents described herein are merelyexemplary and that equivalents of such are known in the art.

The term “construct” or “DNA construct” refers to an engineered fragmentof DNA containing genes and other functional elements. The construct maybe linear or circular. Examples of DNA constructs include plasmids,cosmids, expression vectors, phagemids, fosmids, and artificialchromosomes such as bacterial artificial chromosomes, yeast artificialchromosomes, and human artificial chromosomes.

The term “protein”, “peptide” and “polypeptide” are used interchangeablyand in their broadest sense refer to a compound of two or more subunitamino acids, amino acid analogs or peptidomimetics. The subunits may belinked by peptide bonds. In another embodiment, the subunit may belinked by other bonds, e.g., ester, ether, etc. A protein or peptidemust contain at least two amino acids and no limitation is placed on themaximum number of amino acids which may comprise a protein's orpeptide's sequence. As used herein the term “amino acid” refers toeither natural and/or unnatural or synthetic amino acids, includingglycine and both the D and L optical isomers, amino acid analogs andpeptidomimetics.

A polynucleotide is composed of a specific sequence of four nucleotidebases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil(U) for thymine when the polynucleotide is RNA. Thus, the term“polynucleotide sequence” is the alphabetical representation of apolynucleotide molecule. This alphabetical representation can be inputinto databases in a computer having a central processing unit and usedfor bioinformatics applications such as functional genomics and homologysearching.

The term “isolated” or “recombinant” as used herein with respect tonucleic acids, such as DNA or RNA, refers to molecules separated fromother DNAs or RNAs, respectively that are present in the natural sourceof the macromolecule as well as polypeptides. The term “isolated orrecombinant nucleic acid” is meant to include nucleic acid fragmentswhich are not naturally occurring as fragments and would not be found inthe natural state. The term “isolated” is also used herein to refer topolynucleotides, polypeptides and proteins that are isolated from othercellular proteins and is meant to encompass both purified andrecombinant polypeptides. In other embodiments, the term “isolated orrecombinant” means separated from constituents, cellular and otherwise,in which the cell, tissue, polynucleotide, peptide, polypeptide,protein, antibody or fragment(s) thereof, which are normally associatedin nature. For example, an isolated cell is a cell that is separatedfrom tissue or cells of dissimilar phenotype or genotype. An isolatedpolynucleotide is separated from the 3′ and 5′ contiguous nucleotideswith which it is normally associated in its native or naturalenvironment, e.g., on the chromosome. As is apparent to those of skillin the art, a non-naturally occurring polynucleotide, peptide,polypeptide, protein, antibody or fragment(s) thereof, does not require“isolation” to distinguish it from its naturally occurring counterpart.In certain embodiments, the polypeptides and/or polynucleotidesdescribed herein are isolated and/or recombinant polypeptides orpolynucleotides.

The term “pro-apoptotic gene” refers to a gene that promotes apoptosisin a cell. Apoptosis is the process of programmed cell death that mayoccur in multicellular organisms. Biochemical events lead tocharacteristic cell changes (morphology) and cell death. These changesinclude blebbing, cell shrinkage, nuclear fragmentation, chromatincondensation, and chromosomal DNA fragmentation. Non-limiting examplesof pro-apoptotic genes include caspases, Bik, Puma, Bim, Bax, Bak, Bid,Bad, Bmf, Noxa, and Hrk.

The term “caspase” refers to proteases that play essential roles inapoptosis (programmed cell death) and necrosis. At least 12 caspaseshave been identified in humans. There are two types of apoptoticcaspases: initiator (apical) caspases and effector (executioner)caspases. Initiator caspases (e.g., CASP2 (Genbank Accession:NM_001224.4), CASP8 (Genbank Accession: NM_001080124.1), CASP9 (GenbankAccession: NM_001229.3), and CASP10 (Genbank Accession: NM_001206524.1))cleave inactive pro-forms of effector caspases, thereby activating them.Effector caspases (e.g., CASP3 (Genbank Accession: NM_004346.3), CASP6(Genbank Accession: NM_001226.3), CASP7 (Genbank Accession:NM_001227.3)) in turn cleave other protein substrates within the cell,to trigger the apoptotic process. The initiation of this cascadereaction is regulated by caspase inhibitors.

The term “p16” refers to Cyclin-dependent kinase inhibitor 2A, (CDKN2A,p16^(Ink4A)) also known as multiple tumor suppressor 1 (MTS-1). It hasroles in tumor suppression and is expressed in senescent cells. The p16gene is regulated by a promoter that is activated in senescent cells. Apromoter is a region of DNA that facilitates or regulates thetranscription of a particular gene. Promoters are located near the genesthey regulate, on the same strand and typically upstream (towards the 5′region of the sense strand).

The term “senescent cell” refers to a cell that has lost the ability todivide. A cell may stop dividing due to a variety of factors such as,for example, cellular damage, DNA damage, and/or toxins. A senescentcell may be a cultured cell or a cell in the body, in vivo.

It is to be inferred without explicit recitation and unless otherwiseintended, that when the present invention relates to a polypeptide,protein, polynucleotide or antibody, an equivalent or a biologicallyequivalent of such is intended within the scope of this invention. Asused herein, the term “biological equivalent thereof” is intended to besynonymous with “equivalent thereof” when referring to a referenceprotein, antibody, polypeptide or nucleic acid, intends those havingminimal homology while still maintaining desired structure orfunctionality. Unless specifically recited herein, it is contemplatedthat any polynucleotide, polypeptide or protein mentioned herein alsoincludes equivalents thereof. For example, an equivalent intends atleast about 70% homology or identity, or alternatively about 80%homology or identity and alternatively, at least about 85%, oralternatively at least about 90%, or alternatively at least about 95% oralternatively 98% percent homology or identity and exhibitssubstantially equivalent biological activity to the reference protein,polypeptide or nucleic acid. In another aspect, the term intends apolynucleotide that hybridizes under conditions of high stringency tothe reference polynucleotide or its complement.

A polynucleotide or polynucleotide region (or a polypeptide orpolypeptide region) having a certain percentage (for example, 80%, 85%,90% or 95%) of “sequence identity” to another sequence means that, whenaligned, that percentage of bases (or amino acids) are the same incomparing the two sequences. The alignment and the percent homology orsequence identity can be determined using software programs known in theart, for example those described in Current Protocols in MolecularBiology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table7.7.1. Preferably, default parameters are used for alignment. Apreferred alignment program is BLAST, using default parameters. Inparticular, preferred programs are BLASTN and BLASTP, using thefollowing default parameters: Genetic code=standard; filter=none;strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+SwissProtein+SPupdate+PIR. Details of these programs can befound at the following Internet address: ncbi.nlm.nih.gov/cgi-bin/BLAST.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology canbe determined by comparing a position in each sequence which may bealigned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then the moleculesare homologous at that position. A degree of homology between sequencesis a function of the number of matching or homologous positions sharedby the sequences. An “unrelated” or “non-homologous” sequence sharesless than 30% identity or alternatively less than 25% identity, lessthan 20% identity, or alternatively less than 10% identity with one ofthe sequences of the present invention.

“Homology” or “identity” or “similarity” can also refer to two nucleicacid molecules that hybridize under stringent conditions to thereference polynucleotide or its complement.

“Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson-Crick base pairing, Hoogstein binding, or inany other sequence-specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming amulti-stranded complex, a single self-hybridizing strand, or anycombination of these. A hybridization reaction may constitute a step ina more extensive process, such as the initiation of a PCR reaction, orthe enzymatic cleavage of a polynucleotide by a ribozyme.

Examples of stringent hybridization conditions include: incubationtemperatures of about 25° C. to about 37° C.; hybridization bufferconcentrations of about 6×SSC to about 10×SSC; formamide concentrationsof about 0% to about 25%; and wash solutions from about 4×SSC to about8×SSC. Examples of moderate hybridization conditions include: incubationtemperatures of about 40° C. to about 50° C.; buffer concentrations ofabout 9×SSC to about 2×SSC; formamide concentrations of about 30% toabout 50%; and wash solutions of about 5×SSC to about 2×SSC. Examples ofhigh stringency conditions include: incubation temperatures of about 55°C. to about 68° C.; buffer concentrations of about 1×SSC to about0.1×SSC; formamide concentrations of about 55% to about 75%; and washsolutions of about 1× SSC, 0.1×SSC, or deionized water. In general,hybridization incubation times are from 5 minutes to 24 hours, with 1,2, or more washing steps, and wash incubation times are about 1, 2, or15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It isunderstood that equivalents of SSC using other buffer systems can beemployed.

The term “essential viral gene” refers to a viral gene required for anessential function of the virus. The function may be integration,replication, or viral structure. Exemplary essential viral genes includegag, pol, and env. Gag encodes the internal structural protein of thevirus. Gag protein is proteolytically processed into the mature proteinsMA (matrix), CA (capsid) and NC (nucleocapsid). The pol gene encodes thereverse transcriptase (RT), which contains DNA polymerase, associatedRNase H and integrase (IN), which mediate replication of the genome.

The term “chemically inducible promoter” refers to a promoter whoseactivity is induced by the presence or absence of a chemical. Oneexample of a chemically inducible promoters is the alcohol dehydrogenaseI promoter, which can be induced by different agricultural alcohol-basedformulations. The tetracycline-regulated promoter systems also providean example of a chemically inducible promoter. The tetracyclinepromoters can function either to activate or repress gene expression inthe presence of tetracycline. Some of the elements of the systemsinclude a tetracycline repressor protein (TetR), a tetracycline operatorsequence (tetO) and a tetracycline transactivator fusion protein (tTA),which is the fusion of TetR and a herpes simplex virus protein 16 (VP16)activation sequence. Also included within this disclosure are steroidregulated promoters such as promoters based on the rat glucocorticoidreceptor, promoters based on the human estrogen receptor, and promotersbased on ecdysone receptors derived from different moth species. Otherchemically inducible promoters are known in the art. It is within theknowledge of the skilled artisan to select a chemically induciblepromoter based on factors such as cell type, deliver system, etc. . . .

A “gene” refers to a polynucleotide containing at least one open readingframe (ORF) that is capable of encoding a particular polypeptide orprotein after being transcribed and translated. Any of thepolynucleotide or polypeptide sequences described herein may be used toidentify larger fragments or full-length coding sequences of the genewith which they are associated. Methods of isolating larger fragmentsequences are known to those of skill in the art.

The term “express” refers to the production of a gene product.

As used herein, “expression” refers to the process by whichpolynucleotides are transcribed into mRNA and/or the process by whichthe transcribed mRNA is subsequently being translated into peptides,polypeptides, or proteins. If the polynucleotide is derived from genomicDNA, expression may include splicing of the mRNA in a eukaryotic cell.

A “gene product” or alternatively a “gene expression product” refers tothe amino acid (e.g., peptide or polypeptide) generated when a gene istranscribed and translated.

“Under transcriptional control” or “regulated by” is a term wellunderstood in the art and indicates that transcription of apolynucleotide sequence, usually a DNA sequence, depends on its beingoperatively linked to an element which contributes to the initiation of,or promotes, transcription.

The term “encode” as it is applied to polynucleotides refers to apolynucleotide which is said to “encode” a polypeptide if, in its nativestate or when manipulated by methods well known to those skilled in theart, it can be transcribed and/or translated to produce the mRNA for thepolypeptide and/or a fragment thereof. The antisense strand is thecomplement of such a nucleic acid, and the encoding sequence can bededuced therefrom.

“Pharmaceutically acceptable carriers” or “carriers” refers to anydiluents, excipients or carriers that may be used in the compositions ofthe invention. Pharmaceutically acceptable carriers include ionexchangers, alumina, aluminum stearate, lecithin, serum proteins, suchas human serum albumin, buffer substances, such as phosphates, glycine,sorbic acid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, Mack Publishing Company, a standard referencetext in this field. They are preferably selected with respect to theintended form of administration, that is, oral tablets, capsules,elixirs, syrups and the like and consistent with conventionalpharmaceutical practices.

“Administration” can be effected in one dose, continuously orintermittently throughout the course of treatment. Methods ofdetermining the most effective means and dosage of administration areknown to those of skill in the art and will vary with the compositionused for therapy, the purpose of the therapy, the target cell beingtreated and the subject being treated. Single or multipleadministrations can be carried out with the dose level and pattern beingselected by the treating physician. Suitable dosage formulations andmethods of administering the agents are known in the art. Route ofadministration can also be determined and method of determining the mosteffective route of administration are known to those of skill in the artand will vary with the composition used for treatment, the purpose ofthe treatment, the health condition or disease stage of the subjectbeing treated and target cell or tissue. Non-limiting examples of routeof administration include oral administration, nasal administration,injection and topical application.

In the case of an in vitro application, in some embodiments theeffective amount will depend on the size and nature of the applicationin question. It will also depend on the nature and sensitivity of the invitro target and the methods in use. The skilled artisan will be able todetermine the effective amount based on these and other considerations.The effective amount may comprise one or more administrations of acomposition depending on the embodiment.

The agents and compositions can be used in the manufacture ofmedicaments and for the treatment of humans and other animals byadministration in accordance with conventional procedures, such as anactive ingredient in pharmaceutical compositions.

An agent of the present invention can be administered for therapy by anysuitable route of administration. It will also be appreciated that thepreferred route will vary with the condition and age of the recipientand the disease being treated.

A “viral vector” is defined as a recombinantly produced virus or viralparticle that comprises a polynucleotide to be delivered into a hostcell, either in vivo, ex vivo or in vitro. Examples of viral vectorsinclude retroviral vectors, lentiviral vectors, adenovirus vectors,adeno-associated virus vectors, alphavirus vectors and the like.Alphavirus vectors, such as Semliki Forest virus-based vectors andSindbis virus-based vectors, have also been developed for use in genetherapy and immunotherapy. See, Schlesinger and Dubensky (1999) Curr.Opin. Biotechnol. 5:434-439 and Ying, et al. (1999) Nat. Med.5(7):823-827.

In aspects where gene transfer is mediated by a lentiviral vector, avector construct or backbone refers to the polynucleotide comprising thelentiviral genome or part thereof, and a therapeutic gene. As usedherein, “lentiviral mediated gene transfer” or “lentiviral transduction”carries the same meaning and refers to the process by which a gene ornucleic acid sequences are stably transferred into the host cell byvirtue of the virus entering the cell and integrating its genome intothe host cell genome. The virus can enter the host cell via its normalmechanism of infection or be modified such that it binds to a differenthost cell surface receptor or ligand to enter the cell. Retrovirusescarry their genetic information in the form of RNA; however, once thevirus infects a cell, the RNA is reverse-transcribed into the DNA formwhich integrates into the genomic DNA of the infected cell. Theintegrated DNA form is called a provirus. As used herein, lentiviralvector refers to a viral particle capable of introducing exogenousnucleic acid into a cell through a viral or viral-like entry mechanism.A “lentiviral vector” is a type of retroviral vector well-known in theart that has certain advantages in transducing nondividing cells ascompared to other retroviral vectors. See, Trono D. (2002) Lentiviralvectors, New York: Spring-Verlag Berlin Heidelberg.

Lentiviral vectors of this invention may be based on or derived fromoncoretroviruses (the sub-group of retroviruses containing MLV), andlentiviruses (the sub-group of retroviruses containing HIV). Examplesinclude ASLV, SNV and RSV all of which have been split into packagingand vector components for lentiviral vector particle production systems.The lentiviral vector particle according to the invention may be basedon a genetically or otherwise (e.g. by specific choice of packaging cellsystem) altered version of a particular retrovirus.

That the vector particle according to the invention is “based on” or hasa “backbone” of a particular retrovirus means that the vector is derivedfrom that particular retrovirus. The genome of the vector particlecomprises components from that retrovirus as a backbone. The vectorparticle contains essential vector components compatible with the RNAgenome, including reverse transcription and integration systems. Usuallythese will include gag and pol proteins derived from the particularretrovirus. Thus, the majority of the structural components of thevector particle will normally be derived from that retrovirus, althoughthey may have been altered genetically or otherwise so as to providedesired useful properties. However, certain structural components and inparticular the env proteins, may originate from a different virus. Thevector host range and cell types infected or transduced can be alteredby using different env genes in the vector particle production system togive the vector particle a different specificity.

A “subject,” “individual” or “patient” is used interchangeably herein,and refers to a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, murines, rats, rabbit,simians, bovines, ovine, porcine, canines, feline, farm animals, sportanimals, pets, equine, and primate, particularly human. Besides beinguseful for human treatment, the present invention is also useful forveterinary treatment of common laboratory mammals, including rodents,rabbits, mice, and the like.

“Host cell” refers not only to the particular subject cell but to theprogeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

An “effective amount” is an amount sufficient to effect beneficial ordesired results. An effective amount can be administered in one or moreadministrations, applications or dosages. Such delivery is dependent ona number of variables including the time period for which the individualdosage unit is to be used, the bioavailability of the therapeutic agent,the route of administration, etc. It is understood, however, thatspecific dose levels of the therapeutic agents of the present inventionfor any particular subject depends upon a variety of factors includingthe activity of the specific compound employed, the age, body weight,general health, sex, and diet of the subject, the time ofadministration, the rate of excretion, the drug combination, and theseverity of the particular disorder being treated and form ofadministration. Treatment dosages generally may be titrated to optimizesafety and efficacy. Typically, dosage-effect relationships from invitro and/or in vivo tests initially can provide useful guidance on theproper doses for patient administration. In general, one will desire toadminister an amount of the compound that is effective to achieve aserum level commensurate with the concentrations found to be effectivein vitro. Determination of these parameters is well within the skill ofthe art. These considerations, as well as effective formulations andadministration procedures are well known in the art and are described instandard textbooks.

The term administration shall include without limitation, administrationby oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous,ICV, intracisternal injection or infusion, subcutaneous injection, orimplant), by inhalation spray nasal, vaginal, rectal, sublingual,urethral (e.g., urethral suppository) or topical routes ofadministration (e.g., gel, ointment, cream, aerosol, etc.) and can beformulated, alone or together, in suitable dosage unit formulationscontaining conventional non-toxic pharmaceutically acceptable carriers,adjuvants, excipients, and vehicles appropriate for each route ofadministration. The invention is not limited by the route ofadministration, the formulation or dosing schedule. Administration mayalso refer to the transfer of genetic material (i.e. polynucleotides) tothe human body. Methods of gene transfer are known in the art and aredescribed herein.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the likeinclude the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above.

DESCRIPTIVE EMBODIMENTS

Senescent cells, but not non-senescent cells, express the p16 gene (acell cycle dependent kinase inhibitor). The expression of the p16 geneis the result of p16 promoter activation in senescent cells. Describedherein are methods and compositions that can be used to increase theexpression of caspases through p16 promoter activation in senescentcells.

Polynucleotides

One aspect relates to a polynucleotide comprising a pro-apoptotic geneand a p16 promoter, wherein the expression of the pro-apoptotic gene isregulated by the p16 promoter or an equivalent thereof. The p16 promotercan be a canonical p16 promoter (See, for e.g. Nature. 2011 Nov. 10;479(7372):232-6 which is incorporated by reference for all purposes) ora non-cononical p16 promoter. The promoter sequence is also disclosed inthe gene sequence for the p16 gene (Genbank Accession Nos: NC_000009.11,GI:224589821, and AF527803.1 which are herein incorporated by referencefor all purposes). The promoter sequence is the sequence upstream(towards the 5′ region of the sense strand) of the transcription startsite of the gene. The promoter sequence of p16 as well as discreetbinding elements in the p16 promoter are disclosed in the art, and askilled artisan would be able to identify an appropriate portion of orall of the p16 promoter to use when practicing the invention. Additionalelements may be inserted into the promoter or as sequences upstream ordownstream of the promoter or coding sequences present on the DNAconstruct. Such elements include enhancers (such as, for example, SV40and the Ig enhancer), and transcription binding elements. Otherenhancer/silencer/insulator sequences are further described in U.S. Pat.Pub. Nos.: 2010/0158879, 2007/0172949, 2003/0229046, 2002/0133838,2002/0066117 and 20040076954, each of which are herein incorporated byreference in their entirety. It is contemplated that the addition ofthese additional elements will fine-tune the activity of the p16promoter to make the promoter stronger, weaker, temporally-regulated,spatially-regulated, tissue-specific, and/or inducible by physical andchemical inducers, for example.

The p16 promoter may be from any species. In certain embodiments, thep16 promoter is from a mammal. In further embodiments, the p16 promotercomprises the human, mouse, or rat p16 promoter, an equivalent thereof,a fragment thereof, or a polynucleotide having at least 80% identity tothe human or mouse p16 promoter. In further embodiments, the p16promoter comprises a polynucleotide having at least 70%, 75%, 80%, 85%,90%, 95%, or 99% identity to the human, mouse, or rat promoter.

In one embodiment, the human p16 promoter comprises SEQ ID NO: 1 or afragment or equivalent thereof. This sequence exemplifies 1000 bpupstream of start codon (ATG). Regulatory elements that contribute tooverexpression of p16 in senescent cells are in the region of −622 to−280 bp. A negative regulatory element, the INK4a transcription silenceelement (ITSE) is at −491 to −485 bp of SEQ ID NO: 1. The GC-rich regionof the p16 promoter from −466 to −451 of SEQ ID NO: 1 is a positivetranscription regulatory element. Deletion of this region showed 91.4%loss of p16 promoter activity in senescent cells (Wang et al.“Characterization of Regulatory Elements on the Promoter Region ofp16INK4a That contribute to Overexpression of p16 in SenescentFibroblasts” J. of Biol. Chem. (2001), Vol. 276, No. 52, pp.48655-48661). Regulatory elements believed to be important within thep16 promoter region include Region A (−507 to −493 bp of SEQ ID NO: 1),Region B (−491 to −485 bp of SEQ ID NO: 1), and Region C (−466 to −450bp of SEQ ID NO: 1). In certain embodiments, the human p16 promoterretains at least 80%, 90%, 95%, or 95% to one or more of Regions A, B,or C.

In a further embodiment, the mouse p16 promoter comprises SEQ ID NO: 2.This sequence exemplifies 2800 bp upstream of start codon (ATG).

The pro-apototic gene may be a pro-apoptotic gene from any species.Preferably, the pro-apototic gene corresponds to a mammalianpro-apototic gene. Non-limiting examples of pro-apoptotic genes includecaspase, Bik, Puma, Bim, Bax, Bak, Bid, Bad, Bmf, Noxa, and Hrk. In oneembodiment, the pro-apoptotic gene is a caspase. In certain embodiments,the caspase is a human caspase. Expression of the caspase in a senescentcell may induce apoptosis of the senescent cell, thereby eliminating thesenescent cell and reducing the risk of disease. Accordingly, in certainembodiments, the caspase is one that is involved in the apoptosispathway of the cell. In a specific embodiment the caspase is caspase 8(See, for e.g., Nature Medicine 11, 797-803 (2005) which is incorporatedby reference for all purposes). In another embodiment, the caspase is achemically dimerizable cyclophilin binding protein caspase (See, fore.g., Nature Medicine 11, 797-803 (2005), which is herein incorporatedby reference in its entirety). In a further embodiment, the caspasecomprises the nucleotide sequence of SEQ ID NO: 3, an equivalentthereof, a fragment thereof, or a polynucleotide having at least 70%,75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 3.

The polynucleotide may include further elements that render thepolynucleotide suitable for expression in a host cell or host senescentcell. Such elements include antibiotic resistance genes, origins ofreplication, and termination sequences, for example. The polynucleotidemay further comprise markers that enable the tracking and expression ofthe polynucleotice in cells. Such markers may include fluorescent labelsand the like. The markers and elements of the polynucleotide may beunder the expression of one promoter or multiple promoters.

Viruses

Described herein is the production of viruses engineered to induceapoptosis in cells that are expressing or containing factors thatactivate the p16 promoter. Activation of the p16 promoter is prevalentin senescent cells but not in non-senescent cells. Thus, it iscontemplated that infecting a cell with virus comprising an apoptosisinducing factor such as a pro-apoptotic gene (i.e. caspase) regulated bythe p16 promoter will induce apoptosis in cells expressing and/orcontaining factors capable of activating the p16 promoter. The promotermay be the standard p16 promoter or an equivalent or comprise SEQ ID NO:1 or 2. The promoter may also contain additional elements that enhanceor reduce the expression of downstream genes.

This disclosure features methods and compositions for expressing a genein a cell. In one aspect, the disclosure features methods of genetherapy to express a gene or protein in a cell, such as a senescent cellof a patient. Gene therapy, including the use of viral vectors asdescribed herein, seeks to transfer new genetic material (e.g.,polynucleotides encoding pro-apoptotic genes or other proteins or abiologically active fragment thereof) to the cells of a patient withresulting therapeutic benefit to the patient.

For in vivo gene therapy, expression vectors encoding the gene ofinterest is administered directly to the patient. The vectors are takenup by the target cells (e.g., senescent cells) and the gene expressed.Recent reviews discussing methods and compositions for use in genetherapy include Eck et al., in Goodman & Gilman's The PharmacologicalBasis of Therapeutics, Ninth Edition, Hardman et al., eds., McGray-Hill,New York, 1996, Chapter 5, pp. 77-101; Wilson (1997) Clin. Exp. Immunol.107 (Suppl. 1):31-32; Wivel et al. (1998) Hematology/Oncology Clinics ofNorth America, Gene Therapy, S. L. Eck, ed., 12(3):483-501; Romano etal. (2000) Stem Cells 18:19-39, and the references cited therein. U.S.Pat. No. 6,080,728 also provides a discussion of a wide variety of genedelivery methods and compositions.

In one embodiment, the virus is capable of infecting non-dividing cells(i.e. a lentivirus). In one aspect, the backbone contains essentialsequences for integration into a target cell genome.

In one aspect, the term “virus” intends a recombinant viral vector thatretains the ability to infect and transduce non-dividing and/orslowly-dividing cells and integrate into the target cell's genome. Inseveral aspects, the vector is derived from or based on a wild-typevirus. In further aspects, the vector is derived from or based on awild-type lentivirus. Examples of such, include without limitation,human immunodeficiency virus (HIV), equine infectious anaemia virus(EIAV), simian immunodeficiency virus (SIV) and feline immunodeficiencyvirus (FIV). Alternatively, it is contemplated that other retrovirus canbe used as a basis for a vector backbone such murine leukemia virus(MLV). It will be evident that a viral vector according to the inventionneed not be confined to the components of a particular virus. The viralvector may comprise components derived from two or more differentviruses, and may also comprise synthetic components. Vector componentscan be manipulated to obtain desired characteristics, such as targetcell specificity.

Adenoviruses are able to transfect a wide variety of cell types,including non-dividing cells. There are more than 50 serotypes ofadenoviruses that are known in the art, but the most commonly usedserotypes for gene therapy are type 2 and type 5. These viruses may berendered replication-defective and genetically-modified to preventunintended spread of the virus. This is normally achieved through thedeletion of the E1 region, deletion of the E1 region along with deletionof either the E2 or E4 region, or deletion of the entire adenovirusgenome except the cis-acting inverted terminal repeats and a packagingsignal (Gardlik et al. (2005) Med. Sci. Monit. 11: RA110-121). Incertain embodiments, the virus is not replication defective.

Retroviruses are also useful as gene therapy vectors and usually (withthe exception of lentiviruses) are not capable of transfectingnon-dividing cells. Accordingly, any appropriate type of retrovirus thatis known in the art may be used, including, but not limited to, HIV,SIV, FIV, EIAV, and Moloney Murine Leukaemia Virus (MoMLV).

In another aspect, the invention features the methods of gene therapythat utilize a lentiviral vector to express pro-apoptotic genes, orother proteins in a patient. Lentiviruses are a type of retroviruseswith the ability to infect both proliferating and quiescent cells. Anexemplary lentiviral vector for use in gene therapy is the HIV-1lentivirus. Previously constructed genetic modifications of lentivirusesinclude the deletion of all protein encoding genes except those of thegag, pol, and rev genes (Moreau-Gaudry et al. (2001) Blood98:2664-2672).

Adeno-associated virus (AAV) vectors can achieve latent infection of abroad range of cell types, exhibiting the desired characteristic ofpersistent expression of a therapeutic gene in a patient. The inventionincludes the use of any appropriate type of adeno-associated virus knownin the art including, but not limited to AAV1, AAV2, AAV3, AAV4, AAV5,and AAV6 (Lee et al. (2005) Biochem. J. 387: 1-15; U.S. PatentPublication 2006/0204519).

Herpes simplex virus (HSV) replicates in epithelial cells, but is ableto stay in a latent state in non-dividing cells such as the midbraindopaminergic neurons. The gene of interest may be inserted into the LATregion of HSV, which is expressed during latency. Other viruses thathave been shown to be useful in gene therapy include parainfluenzaviruses, poxviruses, and alphaviruses, including Semliki forest virus,Sinbis virus, and Venezuelan equine encephalitis virus (Kennedy (1997)Brain 120: 1245-1259).

With regard to the structural genes gag, pol and env themselves, gagencodes the internal structural protein of the virus. Gag protein isproteolytically processed into the mature proteins MA (matrix), CA(capsid) and NC (nucleocapsid). The pol gene encodes the reversetranscriptase (RT), which contains DNA polymerase, associated RNase Hand integrase (IN), which mediate replication of the genome.

For the production of viral vector particles, the vector RNA genome isexpressed from a DNA construct encoding it in a host cell. Thecomponents of the particles not encoded by the vector genome areprovided in trans by additional nucleic acid sequences (the “packagingsystem”, which usually includes either or both of the gag/pol and envgenes) expressed in the host cell. The set of sequences required for theproduction of the viral vector particles may be introduced into the hostcell by transient transfection, or they may be integrated into the hostcell genome, or they may be provided in a mixture of ways. Thetechniques involved are known to those skilled in the art.

Retroviral vectors for use in this invention include, but are notlimited to Invitrogen's pLenti series versions 4, 6, and 6.2 “ViraPower”system. Manufactured by Lentigen Corp.; pHIV-7-GFP, lab generated andused by the City of Hope Research Institute; “Lenti-X” lentiviralvector, pLVX, manufactured by Clontech; pLKO.1-puro, manufactured bySigma-Aldrich; pLemiR, manufactured by Open Biosystems; and pLV, labgenerated and used by Charité Medical School, Institute of Virology(CBF), Berlin, Germany.

Also provided are conditionally replicating viruses in which regulatoryelements responsible for the expression of essential viral genes arereplaced by the p16 promoter as described herein to ensure that theviruses will replicate and subsequently kill only senescent cells.

Suitable viruses may include, by way of non-limiting examples,Adeno-associated virus, Australian bat lyssavirus, BK polyomavirus,Banna virus, Barmah forest virus, Bunyamwera virus, Bunyavirus LaCrosse, Bunyavirus snowshoe hare, Cercopithecine herpesvirus,Cercopithecine herpesvirus, Chandipura virus, Chikungunya virus, Cowpoxvirus, Coxsackievirus, Crimean-Congo hemorrhagic fever virus, Denguevirus, Dhori virus, Dugbe virus, Duvenhage virus, Eastern equineencephalitis virus, Ebolavirus, Echovirus, Encephalomyocarditis virus,Epstein-Barr virus, European bat lyssavirus, GB virus C, Hantaan virus,Hendra virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus,Hepatitis E virus, Hepatitis delta virus, Human herpesvirus 1, Horsepoxvirus, Human adenovirus, Human astrovirus, Human coronavirus, Humancytomegalovirus, Human enterovirus 68, 70, Human herpesvirus 2, Humanherpesvirus 6, Human herpesvirus 7, Human herpesvirus 8, Humanimmunodeficiency virus, Human papillomavirus, Human parainfluenza, Humanparvovirus B19, Human respiratory syncytial virus, Human respiratorysyncytial virus, Human respiratory syncytial virus, Human SARScoronavirus, Human spumaretrovirus, Human T-lymphotropic virus, Humantorovirus, Influenza A virus, Influenza B virus, Influenza C virus,Isfahan virus, JC polyomavirus, Japanese encephalitis virus, Juninarenavirus, KI Polyomavirus, Kunjin virus, Lagos bat virus, LakeVictoria marburgvirus, Langat virus, Lassa virus, Lordsdale virus,Louping ill virus, Lymphocytic choriomeningitis virus, Machupo virus,Mayaro virus, Measles virus, Mengo encephalomyocarditis virus, Merkelcell polyomavirus, Mokola virus, Molluscum contagiosum virus, Monkeypoxvirus, Mumps virus, Murray valley encephalitis virus, New York virus,Nipah virus, Norwalk virus, O'nyong-nyong virus, Orf virus, Oropouchevirus, Pichinde virus, Poliovirus, Punta toro phlebovirus, Puumalavirus, Rabies virus, Rift valley fever virus, Ross river virus,Rotavirus A, Rotavirus B, Rotavirus C, Rubella virus, Sagiyama virus,Sandfly fever sicilian virus, Sapporo virus, Semliki forest virus, Seoulvirus, Simian foamy virus, Simian virus 5, Sindbis virus, Southamptonvirus, St. louis encephalitis virus, Tick-borne powassan virus, Torqueteno virus, Toscana virus, Uukuniemi virus, Vaccinia virus,Varicella-zoster virus, Variola virus, Venezuelan equine encephalitisvirus, Vesicular stomatitis virus, Western equine encephalitis virus, WUpolyomavirus, West Nile virus, Yaba monkey tumor virus, Yaba-likedisease virus, Yellow fever virus.

While the list of virus above provides names of individual virus types,it is contemplated that any serotype from a given virus would besuitable for use. For instance there are more than 100 types ofpapillomaviruses and a dozen or more serotypes of adenovirus. Alsouseful are viruses not known to infect human cells but engineered insuch fashion that they gain the ability to infect and or replicatelytically in human cells.

Viruses can be expressed in cells by methods common in the art. Methodsof introduction of a virus in the cell include common methods ofinfection or transfection methods. When the methods are practiced inhumans, it is preferred that the virus is introduced into the cell byinfection.

Gene Transfer of Polypeptides

Exemplary non-viral vectors for delivering nucleic acid include nakedDNA; DNA complexed with cationic lipids, alone or in combination withcationic polymers; anionic and cationic liposomes; DNA-protein complexesand particles comprising DNA condensed with cationic polymers such asheterogeneous polylysine, defined-length oligopeptides, and polyethyleneimine, in some cases contained in liposomes; and the use of ternarycomplexes comprising a virus and polylysine-DNA. In vivo DNA-mediatedgene transfer into a variety of different target sites has been studiedextensively. Naked DNA may be administered using an injection, a genegun, or electroporation. Naked DNA can provide long-term expression inmuscle. See Wolff et al. (1992) Human Mol. Genet. 1:363-369; Wolff etal. (1990) Science 247: 465-1468. DNA-mediated gene transfer has alsobeen characterized in liver, heart, lung, brain and endothelial cells.See Zhu et al. (1993) Science 261:209-211; Nabel et al. (1989) Science244:1342-1344. DNA for gene transfer also may be used in associationwith various cationic lipids, polycations and other conjugatingsubstances. See Przybylska et al. (2004) J. Gene Med. 6:85-92; Svahn etal. (2004) J. Gene Med. 6:S36-S44.

Methods of gene therapy using cationic liposomes are also well known inthe art. Exemplary cationic liposomes for use in this invention areDOTMA, DOPE, DOSPA, DOTAP, DC-Chol, Lipid GL-67.TM., and EDMPC. Theseliposomes may be used in vivo or ex vivo to encapsulate a vector fordelivery into target cells (e.g., neurons or pluripotent stem cells).

Pseudotyped Viral Particles

This invention further provides a method for producing a pseudotypedviral particle, comprising transducing a packaging cell line with theviral vector as described above, under conditions suitable to packagethe viral vector. Such conditions are known in the art and brieflydescribed herein. The pseudotyped viral particle can be isolated fromthe cell supernatant, using methods known to those of skill in the art,e.g., centrifugation. Such isolated particles are further provided bythis invention.

This invention further provides the isolated pseudotyped viral particleproduced by this method. The pseudotyped viral particle comprises apolypeptide comprising a pro-apoptotic gene and a p16 promoter, whereinthe expression of the pro-apoptotic gene is regulated by the p16promoter or an equivalent thereof or embodiments of this polypeptide asdescribed herein.

Yet further provided is an isolated cell or population of cells,comprising a polypeptide, viral particle, or viral vector as describedherein. In one aspect, the isolated host cell is a packaging cell line.

Methods of the Invention

Aspects include methods for expressing a pro-apoptotic gene (i.e.caspase) in a senescent cell comprising administering to the cell apolynucleotide, viral particle, viral vector, or pharmaceuticalcomposition as described herein. The expression of proteins in a cellcan be determined by standard techniques known in the art. By way ofexample, such methods include, Southern blot analysis, western blotanalysis, reverse-transcriptase PCR, real-time PCR, and the like. Thecell may be one in which the p16 promoter is activated. However, it isnot necessary to infect only cells with an activated p16 promoter sinceit is believed that the viruses described herein will only be active andreplicate in cells in which the p16 promoter is activated.

Another method aspect relates to a method for inducing apoptosis in asenescent cell comprising administering to the cell a polynucleotide,viral particle, viral vector, or pharmaceutical composition as describedherein. The cell may be a cell in cell culture or a cell in vivo, in themammalian body.

Various assays can be used to determine the effectiveness of suchmethods. Assays that determine the level of cell death can be used andinclude, for example a TUNEL (Terminal deoxynucleotidyl transferase dUTPnick end labeling) assay. A TUNEL assay is a method for detecting DNAfragmentation by labeling the terminal end of nucleic acids. Anothermethod of determing the level of cell death in cells is throughpropidium iodide labeling of cells. Propidium iodide is a DNA stain thatcan differentiate necrotic, apoptotic and normal cells. Live cells anddead cells can also be differentiated using trypan blue, a stain thatcolors dead tissues or dead cells blue. Other viability assays includebut are not limited to ATP test, clonogenic assay, evans blue, flowcytometry, formazan-based assays, lactate dehydrogenase, methyl violet,and resazurin.

Pharmaceutical Compositions

The polypeptides, viral particles, and viral vectors as described hereinmay be combined with a carrier or a pharmaceutically acceptable carriersuitable for use of the compositions in the methods disclosed herein.

Compositions of the invention may be conventionally administeredparenterally, by injection, for example, intravenously, subcutaneously,or intramuscularly. Additional formulations which are suitable for othermodes of administration include oral formulations. Oral formulationsinclude such normally employed excipients such as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate and the like. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders and contain about10% to about 95% of active ingredient, preferably about 25% to about70%. In certain embodiments, a composition may be inhaled (e.g., U.S.Pat. No. 6,651,655, which is specifically incorporated by reference inits entirety). In the case of viral particles, the term administrationincludes the infection of a cell with the viral particle. Polypeptidesand viral vectors of the disclosure may also be administered by variousmethods of gene transfer as described herein.

Typically, compositions of the invention are administered in a mannercompatible with the dosage formulation, and in such amount as will betherapeutically effective. The quantity to be administered depends onthe subject to be treated. Precise amounts of active ingredient requiredto be administered depend on the judgment of the practitioner.

The phrases “pharmaceutically acceptable” or “pharmacologicallyacceptable” refer to molecular entities and compositions that do notproduce an adverse, allergic, or other untoward reaction whenadministered to an animal, or human. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like. The use of such media and agents forpharmaceutical active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredients, its use in therapeutic compositions is contemplated.

The carrier may be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid poly(ethylene glycol), and the like), suitablemixtures thereof, and vegetable oils. The proper fluidity can bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersion,and by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

An effective amount of therapeutic composition is determined based onthe intended goal. The term “unit dose” or “dosage” refers to physicallydiscrete units suitable for use in a subject, each unit containing apredetermined quantity of the composition calculated to produce thedesired responses discussed above in association with itsadministration, i.e., the appropriate route and regimen. The quantity tobe administered, both according to number of treatments and unit dose,depends on the result and/or protection desired. Precise amounts of thecomposition also depend on the judgment of the practitioner and arepeculiar to each individual. Factors affecting dose include physical andclinical state of the subject, route of administration, intended goal oftreatment (alleviation of symptoms versus cure), and potency, stability,and toxicity of the particular composition. Upon formulation, solutionswill be administered in a manner compatible with the dosage formulationand in such amount as is therapeutically or prophylactically effective.The formulations are easily administered in a variety of dosage forms,such as the type of injectable solutions described above.

Treatment of Disease

It is contemplated that the methods described herein are useful in thetreatment of diseases such as, for example, Diabetes, Heart Conditions,Obesity, Alzheimer's Disease, Dementia, Parkinson's Disease, Arthritis,Osteoarthritis, Osteoporosis, Loss of Vision, Loss of Hearing, Breathingproblems, Coronary artery disease, Atherosclerosis, Melanocytic Nevi,Cancer, Asthma, Chronic Obstructive Pulmonary Disease, Fracture,Frailty, Hepatitis, Kidney Diseases, Muscle Fatigue, Skin conditions,and Hair loss.

EXAMPLES

Lentiviral production: To test whether senescent cells can be eliminatedusing a conditionally replicating virus, a canonical p16 promoter iscloned into a lentiviral vector. Caspase 8 is cloned downstream of thep16 promoter. Optionally, the promoters of essential lentiviral genessuch as gag, pol and env are also replaced by the p16 promoter and/or achemically inducible promoter. As a control, a lentiviral vector with afluorescent marker instead of the caspase 8 gene is also constructed.The lentiviral constructs can then be transfected into a packaging cellline such as, for example, 293T cells. Transfection of the lentiviralconstructs can be done using standard lipofectamine transfectiontechniques. If necessary, activation factors or chemical inducers areadded to the cells to induce replication of the virus in the packagingcells. After culture of the cells for 24 hours or more, thevirus-containing culture medium is removed from the cells and filtersterilized. The control (p16-fluorescent construct) and experimental(p16-caspase 8) virus are used in in vitro and in vivo experiments.

In vitro apoptosis of senescent cells: Mammalian cells are plated on aculture dish and passaged until replicative senescence is achieved in amajority of the cells. Senescence can be measured by, for example, abeta-galactosidase assay. Virus is added to the cell culture media ofthe senescent cells. The virus is removed after 24 hours, and fresh cellculture media is added to the cells. Optionally, more virus can be addedafter a 12-hour recovery. In parallel, control virus is added to adifferent dish of senescent cells. At 12, 24, 48, and 72 hours afterremoval of the virus, cells are harvested and real-time PCR is performedto test for the expression of caspase 8. In parallel, a TUNEL assay andcaspase 8 immunofluorescence is performed to determine the level ofapoptosis. The level of apoptosis with virus containing the p16-caspase8 construct is compared to the level of apoptosis with thep16-fluorescent construct. It is contemplated that an increase inapoptosis will be observed in cells transfected with the caspase8-containing construct.

In vivo apoptosis of senescent cells: Groups of mice can be inoculatedwith either control or experimental virus at the same titer in a volumeof 100 μl. Animals of two groups can be inoculated subcutaneously in thescruff of the neck. Two, five, seven, fourteen, and 20 days afterinoculation of the mice, individual mice can be sacrificed and testedfor induction of apoptosis in senescent cells, serologic testing, andhistologic examination. Serum samples can be obtained and submitted forserologic testing for antibody testing against a panel of lentiviralantigens. All animals may then be examined for gross lesions, andabnormal tissues can be fixed in formalin, embedded, sectioned atfive-micron thickness, and stained with hematoxylin and eosin.Immunofluoresence and immunohistochemistry can be performed for p16 (tomark senescent cells) and caspase 8 (to mark apoptotic cells) on thesections. The level of apoptosis is compared between the two groups. Itis contemplated that more apoptosis is observed in the senescent cellsof the experimental group versus the control group.

It should be understood that although the present invention has beenspecifically disclosed by preferred embodiments and optional features,modification, improvement and variation of the inventions embodiedtherein herein disclosed may be resorted to by those skilled in the art,and that such modifications, improvements and variations are consideredto be within the scope of this invention. The materials, methods, andexamples provided here are representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of theinvention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

All publications, patent applications, patents, and other referencesmentioned herein are expressly incorporated by reference in theirentirety, to the same extent as if each were incorporated by referenceindividually. In case of conflict, the present specification, includingdefinitions, will control.

What is claimed is:
 1. A composition comprising a conditionallyreplicating virus in a pharmaceutically compatible excipient, whereinthe conditionally replicating virus has a recombinant genome constructin which a replication gene for the virus is placed undertranscriptional control of a heterologous promoter, wherein theheterologous promoter is the p16 promoter, wherein when theconditionally replicating virus is applied to a mixed cell populationcomprising target cells that express p16, the p16 promoter causes thevirus to replicate preferentially in the target cells, thereby causinglysis of the target cells and selectively depleting them from the cellpopulation.
 2. The composition of claim 1, wherein the genome constructof the conditionally replicating virus comprises more than oneheterologous promoter.
 3. The composition of claim 1, wherein the genomeconstruct of the conditionally replicating virus comprises a replicationgene for the virus placed under transcriptional control of a chemicallyinducible promoter.
 4. The composition of claim 1, wherein replicationof the virus is tissue specific.
 5. The composition of claim 1, whereinthe conditionally replicating virus is an adenovirus.
 6. The compositionof claim 1, wherein the conditionally replicating virus is a lentivirus,and the replication gene under transcriptional control of the p16promoter is selected from gag, pol and env.
 7. The composition of claim1, wherein the nucleotide sequence of the p16 promoter comprises SEQ. IDNO:1.
 8. The composition of claim 1, wherein the nucleotide sequence ofthe p16 promoter comprises SEQ. ID NO:2.
 9. The composition of claim 1,wherein the genome of the conditionally replicating virus furthercomprises a caspase gene under transcriptional control of the p16promoter.
 10. A method of selectively depleting senescent cells from amixed cell population or tissue, comprising combining the tissue with acomposition according to claim 1 such that the conditionally replicatingvirus replicates in the senescent cells in the mixed cell population ortissue.